Methods useful in studying or modulating skin or hair pigmentation, plant extracts for use in compositions and cosmetic care method

ABSTRACT

The invention relates to methods useful in studying or modulating skin or hair pigmentation and to the use of plant extracts in compositions as well as to methods comprising the topical use of such compositions to reduce or to enhance skin or hair pigmentation.

The present invention relates to methods useful in studying ormodulating skin or hair pigmentation, to the use of plant extracts, asactive agents modulating melanin skin or hair pigmentation for thepreparation of compositions and to cosmetic care method.

In humans, pigmentation results from the synthesis and distribution ofmelanin pigments in the skin, the hair follicles or the eyes.Pigmentation is genetically predefined, but it is regulated by numerousinternal or external factors. The melanins produced by melanocytes andalso the number of melanocytes, their tyrosinase activity and theirability to export melanins to keratinocytes, and the size of themelanosomes which contain melanin granules, will condition the colour ofhuman skin. For each individual, the colour of the skin varies mainlyaccording to how much or how little irradiation it receives fromultraviolet (UV) rays. In other words, for each individual, there is abasic skin pigmentation when said individual is subjected to the weakestUV irradiation, corresponding to his or her lightest skin colour, and amore intense skin pigmentation if he or she receives a stronger UVirradiation, ranging up to the maximum pigmentation corresponding to hisor her darkest skin colour when subjected to sustained exposure tointense UV irradiation.

Furthermore, as is well known, a very large genetic diversity withregard to skin pigmentation exists in the worldwide population. Thus,according to populations, the skin colour corresponding to the basicpigmentation defined above has a darker or lighter shade which liesbetween the two extremes: very light and very dark. Also according topopulations, the difference in skin shade between the basic pigmentationand the maximum pigmentation is more or less great. Thus, it is wellknown that individuals belonging to certain populations with light skin(basic pigmentation) react rapidly and/or considerably to the action ofUV radiation and can therefore readily exhibit skin with a dark shade,even when these individuals have not intentionally exposed themselves tothe sun for a prolonged period.

Moreover, individuals experience the appearance on their skin, inparticular on the face or the hands, of areas and/or spots which aredarker and/or more coloured, conferring a colour heterogeneity on theskin. These spots are due to a large concentration of melanin in theepidermal keratinocytes resulting from an exacerbated melanogenicactivity in the melanocytes.

The mechanism of formation of skin pigmentation involves the synthesisof melanins. This mechanism is particularly complex and involves,schematically, the following main steps:

Tyrosine→Dopa→Dopaquinone→Dopachrome→Melanins

Tyrosinase and tyrosinase-related-protein 1 (TRP-1) are essentialenzymes involved in this series of reactions. They catalyse inparticular the reaction for conversion of tyrosine to Dopa(dihydroxyphenylalanine) and the reaction for conversion of Dopa toDopaquinone, resulting in the formation of melanin pigments.

A molecule is recognized to be a depigmenting molecule if it actsdirectly on the epidermal melanocytes by inhibiting the activity ofthese cells and/or if it blocks one of the steps of melanin biosynthesisor else if it degrades the melanin formed. This is in particular thecase when the molecule inhibits one of the enzymes involved inmelanogenesis or when it reacts with the chemical compounds of themelanin synthesis chain.

Known depigmenting substances are in particular hydroquinone andderivatives thereof, kojic acid, arbutin, iminophenols, the combinationof carnitine and quinone, aminophenol amide derivatives, andbenzothiazole derivatives. These substances can have certain drawbacks.They can be unstable, can require use at high concentrations, can lackspecificity with regard to their method of action, or can have acytotoxic or irritant capacity.

On the other hand, a molecule is recognized to be pigmenting molecule byits stimulating activity on melanogenesis in the melanocytes present inthe skin or the hair follicles, and thus making possible to promote thepigmentation of the skin or hair as well as to treat disorders of thepigmentation of the skin and hair, more particularly by promoting thebiosynthesis of melanin.

Known pigmenting substances are in particular adenylate cyclaseactivators such as forskoline (Coleus forskholii extract) or Tephrosiapurpurea extract.

Melanocytes are neural crest-derived cells distributed along the basallayer of the epidermis and hair bulb. These cells synthesize melaninpigment through a process called melanogenesis within uniquemelanocyte-specific organelles called melanosomes. For skin and hair tobecome pigmented this melanin must be transferred from melanocytes toadjacent keratinocytes. Mammalian skin eyes, and hair/wool/fur colour isdetermined by melanin quantity and quality (brown/black eumelanin orred/yellow pheomelanin) in a process that involves a large number ofsteps regulated at multiple control points by a range of differentmolecules and compounds. The last couple of decades have seen muchprogress in our understanding of the molecular control of melaninsynthesis and melanosome biogenesis/maturation.

However the knowledge on how melanin transfer is controlled would have aprimary clinical/cosmetic interest as this process ultimately controlsthe level of pigmentation perceived at the skin surface and along thehair fiber.

The inventors have shown the mechanism that controls melanin transferfrom human cutaneous melanocytes to human cutaneous keratinocytesinvolving the action of myosin X (Myo-X), which regulates the transferof melanin from melanocytes to keratinocytes.

The involvement of Myo-X in melanocyte biology (e.g, melanin transfer)has not been reported so far. While much is known about the regulationof melanogenesis (i.e. melanin synthesis) in melanocytes, the knowledgeof how melanosomes transfer from melanocytes to keratinocytes is stillvery limited. In addition to a role for cyto-phagocytosis, there isincreasing evidence to support melanosome transfer via filopodia whichcan interact with keratinocyte plasma membrane (Scott et al., 2002,Singh et al., 2008). Importantly structures consistent with filopodiaarising from the sides and tips of melanocyte dendrites and whichcontain melanosomes within their lumina, have already been reported inhuman skin in situ as well as in vitro (Scott et al., 2002).

During studies on the biology of melanin transfer the present inventorsinvestigated the potential role of a Myo-X in this process. In thisapplication a novel mechanism that controls melanin transfer from humancutaneous melanocytes to human cutaneous keratinocytes is described.

This mechanism involves the action of Myo-X. Myo-X regulates theproduction of nanotubules (called filopodia) on melanocytes, which actas intercellular conduits for melanin transfer. To provide evidence thatMyo-X regulates melanin transfer we used gene-silencing technology toknockdown its expression in melanocytes. Lack of Myo-X resulted in theloss of melanocyte filopodia and the concomitant inhibition ofmelanosome transfer to keratinocytes.

The present inventors have examined how filopodia production isregulated and have observed that actin-based motor proteins of themyosin superfamily may be involved. Specifically, Myosin-X (Myo-X)—a 240KDa vertebrate-specific MyTH4-FERM myosin—is critical for filopodiaformation (Sousa and Cheney 2005). It functions as part of a filopodialtip complex and/or by transporting molecules required for filopodiaformation (Bohil et al., 2006).

In this application it is reported for the first time that Myo-X cancontrol melanin transfer between human melanocytes and humankeratinocytes. Myo-X and molecules based on its structure and action canbe used as a novel regulation of mammalian skin and hair pigmentation.

According to a first aspect, the present invention provides a method ofassessing the ability of a substance to modulate the transfer of melaninfrom a melanocyte to a keranocyte, the method comprising the step of:

-   -   a)—providing a test substance;

b)—providing a cell expressing Myo-X;

-   -   c)—determining the ability of the test substance to modulate the        expression or the activation of Myosin-X (Myo-X).

“Modulating” means that the active agent is able either to increase theexpression or the activity of Myo-X or to decrease the expression or theactivity of Myo-X in humans, this active agent acting either directly onMyo-X or indirectly, i.e. on an upstream target in the Myo-X metabolicpathway.

Preferably the cell expressing Myo-X is a melanocyte or a keratinocyte,more preferably a melanocyte.

The method of assessing the ability of a substance to modulate thetransfer of melanin according to the present invention comprisescomparing the expression or activation of Myo-X in a cell treated with atest substance with the expression or the activation of Myo-X in acontrol cell.

Determining said ability includes:

-   -   measuring the amount of Myo-X protein in or on the cell;    -   determining the amount of a precursor in the synthesis of Myo-X        protein; or    -   measuring the melanosome transfer level in the cell.

Methods for determining the expression of Myo-X include measuring theamount of Myo-X protein in or on the cell, or determining the amount ofa precursor in the synthesis of Myo-X protein, for example mRNA whichcodes for Myo-X. Techniques required to determine the level ofexpression of Myo-X protein or Myo-X mRNA levels would be apparent tothe person skilled in the art. Techniques to determine protein levelsmay involve immunostaining, ELISA, two-dimensional electrophoresis ormass-spectrometric methods. Techniques to determine mRNA levels mightinvolve northern blotting, reverse transcriptase PCR, quantitative PCR,microarrays or Affymetrix® chips.

The level of expression or activation of Myo-X may be determined by manymethods apparent to the person skilled in the art. In embodiments of thepresent invention the expression may be determined usingimmunofluorescence, western blotting or by determining the level ofMyo-X mRNA. As mentioned above, other suitable methods would be apparentto the person skilled in the art.

Methods for determining the activity of Myo-X include measuring themelanosome transfer level in a cell. Techniques required to determinesuch level may involve the quantitative analysis of the melanin transferto keratinocytes and would be apparent to the person skilled in the art.

Techniques to determine melanin transfer level may involve the use ofgp100 as molecular tracker such as describes by Singh S K et al., Exp.Dermatol. 17, 5, 418-426 or any suitable methods apparent to the personskilled in the art.

According to other aspects, the present invention provides a method ofselecting an active substance capable to enhance skin or hairpigmentation comprising:

-   -   assessing the ability of said substance to modulate the transfer        of melanin according to steps a) to c) of the method of claim 1,    -   selecting the substance capable to increase the expression or        the activation of Myo-X and a method of selecting a substance        capable to reduce skin or hair pigmentation comprising:    -   assessing the ability of said substance to modulate the transfer        of melanin according to steps a) to c) of the method of claim 1,    -   selecting the substance capable to decrease the expression or        the activation of Myo-X.

In the sense of the present invention, “skin or hair pigmentation” hasto be understood as “skin or hair pigmentation in humans”.

According to the present invention, the assessed or test substance is aplant extract. The plant extract selected by the method of the inventionas being capable to increase the expression or the activation of Myo-Xis chosen in the group consisting of an extract of soybean, preferably asoy seed extract, more preferably a soy seed pericarp extract and theplant extract selected by the method of the invention as being capableto decrease the expression or the activation of Myo-X is chosen in thegroup consisting of: an extract of Artocarpus genus plant, preferably aArtocarpus heterophyllus extract, more preferably a Artocarpusheterophyllus seed extract; an extract of Cyathea genus plant,preferably a Cyathea cumingii extract more preferably a Cyathea cumingiileaf extract; an extract of Secale genus plant, preferably a Secalecereale extract, more preferably a Secale cereale seed extract; anextract of Thalassiosira genus plant, preferably the secretion ofThalassiosira pseudonana and an extract of Buddleja genus plant,preferably a Buddleja axillaris extract, more preferably a Buddlejaaxillaris leaf extract.

According to another aspect, the present invention provides the use ofat least one of the above plant extract in a cosmetic composition, or inthe preparation of a cosmetic composition as an active agent modulatingthe expression or the activity of Myo-X, said composition being intendedto modulate skin or hair pigmentation.

According to the present invention, the plant extract can be prepared byvarious extraction processes known to a person skilled in the art.

The extraction is preferably performed by placing the plant materialselected in contact with a polar solvent or a mixture of polar solvents.According to this invention, the term “polar solvent” means that thesolvent has a polarity index value equal to or greater than a value of4. The polarity index is a quantity calculated on the basis ofthermodynamic quantities (of solubility and change in state) indicatingthe more or less polar nature of a molecule.

Reference can be made, for the solvent polarity indices, to the articleof L. R. SNYDER: Classification of the solvent properties of commonliquids; Journal of Chromatography, 92 (1974), 223-230, which isincluded by reference to this application.

The polar solvent is advantageously chosen from water, C1-C4 alcohols,such as ethanol, glycols, ethylene glycol, glycerol, butyleneglycol andpropyleneglycol and mixtures thereof.

Preferably, the extraction is performed by using a hydro-alcoholicmixture, in particular a water-ethanol mixture, and preferably awater-ethanol mixture, advantageously in a 50/50 (v/v) ratio.

According to the present invention, the at least plant extract capableto modulate the expression or the activity of Myo-X is chosen in thegroup consisting of an extract of Artocarpus genus plant, preferably aArtocarpus heterophyllus extract, more preferably a Artocarpusheterophyllus seed extract; an extract of Cyathea genus plant,preferably a Cyathea cumingii extract, more preferably a Cyatheacumingii leaf extract; an extract of Secale genus plant, preferably aSecale cereale extract more preferably a Secale cereale seed extract; anextract of Thalassiosira genus plant, preferably the secretion ofThalassiosira pseudonana; an extract of Soybean genus plant, preferablya soy seed extract, more preferably a soy seed pericarp extract; anextract of Buddleja genus plant, preferably a Buddleja axillaris extractmore preferably a Buddleja axillaris leaf extract.

In particular, an extract of Artocarpus genus plant, preferably aArtocarpus heterophyllus extract, more preferably a Artocarpusheterophyllus seed extract; an extract of Cyathea genus plant,preferably a Cyathea cumingii extract more preferably a Cyathea cumingiileaf extract; an extract of Secale genus plant, preferably a Secalecereale extract, more preferably a Secale cereale seed extract; anextract of Thalassiosira genus plant, preferably the secretion ofThalassiosira pseudonana; an extract of Buddleja genus plant, preferablya Buddleja axillaris extract, more preferably a Buddleja axillaris leafextract are active agents decreasing the expression or the activity ofMyo-X and an extract of soybean, preferably a soy seed extract, morepreferably a soy seed pericarp extract, is an active agent increasingthe expression or the activity of Myo-X.

Another object of the present invention is to provide at least a plantextract chosen in the group consisting of an extract of Cyathea genusplant, preferably a Cyathea cumingii extract more preferably a Cyatheacumingii leaf extract; an extract of Secale genus plant, preferably aSecale cereale extract, more preferably a Secale cereale seed extract;an extract of Thalassiosira genus plant, preferably the secretion ofThalassiosira pseudonana; an extract of soybean, preferably a soy seedextract, more preferably a soy seed pericarp extract, for the use in acosmetic composition, or in the preparation of the cosmetic compositionas an active agent modulating the skin or hair pigmentation inparticular, an extract of Cyathea genus plant, preferably a Cyatheacumingii extract more preferably a Cyathea cumingii leaf extract; anextract of Secale genus plant, preferably a Secale cereale extract, morepreferably a Secale cereale seed extract; an extract of Thalassiosiragenus plant, preferably the secretion of Thalassiosira pseudonana; areactive agents for use to reduce the skin or hair pigmentation and anextract of soybean, preferably a soy seed extract, more preferably a soyseed pericarp extract is an active agent for use to enhance the skin orhair pigmentation.

According to another aspect of the present invention there is provided acosmetic care method comprising the topical use of a cosmeticcomposition comprising at least one plant extract chosen in the groupconsisting of an extract of Cyathea genus plant, preferably a Cyatheacumingii extract more preferably a Cyathea cumingii leaf extract; anextract of Secale genus plant, preferably a Secale cereale extract, morepreferably a Secale cereale seed extract; an extract of Thalassiosiragenus plant, preferably the secretion of Thalassiosira pseudonana; anextract of soybean, preferably a soy seed extract, more preferably a soyseed pericarp extract, in order to reduce or to enhance skin or hairpigmentation.

According to a further aspect of the present invention there is provideda cosmetic care method comprising the topical use of a cosmeticcomposition comprising at least one plant extract, obtainable by themethods of the invention as active agent modulating the expression orthe activity of Myo-X in order to reduce or to enhance skin or hairpigmentation.

In particular, an extract of Cyathea genus plant, preferably a Cyatheacumingii extract more preferably a Cyathea cumingii leaf extract; anextract of Secale genus plant, preferably a Secale cereale extract, morepreferably a Secale cereale seed extract; an extract of Thalassiosiragenus plant, preferably the secretion of Thalassiosira pseudonana areactive agents reducing skin pigmentation and an extract of soybean,preferably a soy seed extract, more preferably a soy seed pericarpextract, are active agents enhancing skin or hair pigmentation.

According to the present invention, the at least plant extract asdescribed above is used to prepare a cosmetic composition or is used asan active agent modulating skin or hair pigmentation in the preparationof cosmetic composition lightening or darkening the skin or hairpigmentation.

According to the present invention, the above plant extracts are used atconcentrations range, expressed in dry weight, between 0.0001% and 10%by weight of the composition of the invention, and preferably between0.01% and 2%.

According to another aspect, the present invention provides a cosmeticcare method wherein a cosmetic composition comprising an activesubstance modulating the expression or the activity of Myo-X istypically used to reduce or to enhance skin or hair pigmentation.

In a preferred embodiment said active substance is a nucleic acid, morepreferably a RNA molecule, capable of modulating the expression of Myo-Xthrough interaction with DNA or RNA coding for Myo-X or a functionalportion thereof.

In particular, said RNA molecule is capable of targeting anti-senseinteraction or RNA interference against mRNA encoding Myo-X.

In one embodiment, said RNA molecule is an mRNA molecule for targetingMyo-X mRNA via RNAi having the sequence 5′-CAGCGGTATAAGAGAAATCAA-3′(SEQID No 1).

It is within the skills of the person skilled in the art to determineother suitable sequences which would exhibit RNAi against Myo-X mRNA.

The cosmetic or pharmaceutical compositions, especially dermatologicalcompositions according to the present invention, have thus a variety ofapplications in cosmetology or dermatology not only where it is desiredto reduce the pigmentation but also where it is desired to enhance thepigmentation.

For example, these depigmenting compositions can be used for obtaining alightening effect of the human skin or for obtaining a homogeneous skincolour. They can also be used in case of appearance on the skin, inparticular on the face or the hands, of areas and/or spots which aredarker and/or more colored, conferring a colour heterogeneity on theskin. These spots are due to a large concentration of melanin in theepidermal keratinocytes resulting from an exacerbated melanogenicactivity in the melanocytes. Said compositions are in particular usedfor treated regional hyperpigmentations due to melanocyte hyperactivity,such as idiopathic melasmas, localized hyperpigmentations due tomelanocyte hyperactivity, such as pigmentary spots referred to as solarlentigo or senile lentigo, accidental hyperpigmentations such asphotosensitization or post-lesional cicatrization, and also certainforms of leucoderma such as vitiligo. In the latter cases, since it isnot possible to repigment the skin, the pigmentation at the periphery ofthe depigmented zones is reduced so as to give the skin a morehomogeneous colour.

In a preferred embodiment of the present invention, cosmeticcompositions comprising at least plant extracts capable of reducing theskin or hair pigmentation (chosen in the group consisting of an extractof Cyathea genus plant, preferably a Cyathea cumingii extract morepreferably a Cyathea cumingii leaf extract; an extract of Secale genusplant, preferably a Secale cereale extract, more preferably a Secalecereale seed extract; and an extract of Thalassiosira genus plant,preferably the secretion of Thalassiosira pseudonana) are used in acosmetic care method for the cosmetic treatment of skin or hairhyperpigmentations including those associated with diseases such as:idiopathic melasmas, lentigo. Said compositions are also used in acosmetic care treatment for cosmetically reducing pigmentation contrastsas being the consequence of a depigmented area surrounded by normally ormore pigmented skin, such in the case of vitiligo.

Depigmenting compositions according to the present invention are alsoused as skin-bleaching agents by certain individuals, in particularthose who are very reactive to UV radiations, so as to lighten theircolouring, in particular that of their face and their hands, in order tomaintain a skin colour which is a slight as possible or at the veryleast to reduce the pigmenting effects of UV rays.

On the other hand, the pigmenting compositions according to the presentinvention can be used as sun products to accelerate or intensifytanning, which apart from the esthetic advantage often sought, enablesthe natural defenses against ultra violet radiations to be strengthenedby increasing the proportions in melanin in the epidermis. Thesecompositions can also be used, for example in a form of creams, to givethe skin a more sunburnt appearance, or else in the form of lotions toprevent and treat the appearance of gray hair.

In a preferred embodiment of the present invention cosmetic compositionscomprising at least plant extracts capable of enhancing the skin or hairpigmentation (chosen in the group consisting of an extract of soybean,preferably a soy seed extract, more preferably a soy seed pericarpextract) are used in a cosmetic care method for the cosmetic treatmentof skin or hair depigmentations including those associated with diseasessuch as: tinea versicolor, pityriasis alba, lupus erythematosus, mycosisfungoides, sarcoidosis, leprosy, syphilis and nevus depigmentosus.

Furthermore the cosmetic compositions according to the present inventionmay include, in addition to the at least plant extract, any other activeagent modulating or not skin or hair pigmentation such as sunscreens andmay also include at least one cosmetically acceptable excipient whichcan be chosen from polymers, surfactants, rheology control agents,fragrances, electrolytes, pH adjusters, antioxidants, preservatives,dyes, mother-of-pearl, pigments and mixtures thereof.

The cosmetic compositions referred to in the present invention areintended for topical use. These compositions can for example be a serum,a lotion, a spray, a foam, a solution, a powder, a pomade, a milk, anemulsion, a tinted cream or a hydrogel, and can be in the form of astick, a patch or a mask.

Moreover, the cosmetic compositions according to the present invention,intended for topical administration, can contain at least one agent forpromoting penetration and diffusion in the cutaneous structures inquestion, such as the agents commonly used in the fields of cosmetologyand dermopharmacy, for example glycerol, propylene glycol, oleic acid oressentials oils, especially menthol and eucalyptol.

Embodiments of the present invention will now be described, by way ofexample only, with reference to the accompanying drawings in which:

FIG. 1 shows the effect of plant extracts of the invention actives onmelanogenesis in normal pigmented human melanocytes.

FIG. 2 shows the effect of plant extracts of the invention onmelanogenesis in moderately-pigmented FM55 melanoma.

FIGS. 3A and 3B show the effect of plant extracts of the invention ontyrosinase activity in pigmented normal human melanocytes.

FIGS. 4A and 4B show the effect of plant extracts of the invention ontyrosinase activity in moderately pigmented FM55 melanoma.

FIGS. 5A and 5B show the effect of plant extracts of the invention, IBMXand Niacinamide on tyrosinase activity in normal human melanocyte andkeratinocyte co-culture.

FIG. 6 shows results of quantitative analysis of melanosome transferafter incubation with the plant extracts of the invention.

FIGS. 7A and 7B show the effect of plant extracts of the invention onMyo-X protein expression by Western Blotting in normal humanmelanocytes.

FIG. 8 shows results of quantitative analysis of melanosome transferafter knockdown of Myo-X expression of epidermal melanocytes inepidermal melanocytes-keratinocytes co-culture.

BACKGROUND

On the results reported below the plant extracts used according to thepresent invention are identified by their respective original codedname, with the following correspondence:

-   -   L-18: Artocarpus heterophyllus seed extract,    -   L-19: Cyathea cumingii leaf extract (polar solvent extraction),    -   L-20: Secale cereale seed extract (polar solvents extraction, in        particular water, followed by an enzymatic hydrolysis),    -   L-21: Secretion of Thalassiosira pseudonana (obtained from a        culture in sea water),    -   L-22: Soy seed pericarp extract (aqueous suspension followed by        an enzymatic hydrolysis),    -   L-23: Buddleja axillaris leaf extract (extraction/concentration,        then lyophilisation/sterilisation).

This report presents data on the effect of various L-18-23 on melanosometransfer in normal human epidermal melanocytes to fully matchedepidermal keratinocytes.

The optimal doses have been standardized for each plant extract activeseparately on epidermal melanocytes and keratinocytes. The effect of allL-18-23 on a) melanin synthesis and b) tyrosinase activity usingmelanocytes cultured alone and cultured together with matchedkeratinocytes (i.e., EM-KC co-cultures) has been assessed. The effectsof the plant extracts actives on melanosome transfer in EM: KCco-culture using gp00 immunolabelling has been evaluated.

Both positive melanocyte modulator (IBMX) and a melanosome transferinhibitor (Niacinamide) were used as control for this study.

In general, L-18, 19, 20, 21, 23 downregulated melanin synthesis,dopa-oxidase activity of tyrosinase, melanosome transfer, and Myo-Xexpression. In fact, L-18 and L-23 were known as depigmentating agentsbut their action on these parameters was not shown so far. By contrast,L-22 was found to upregulate said parameters.

Materials & Methods

Stimulation of Melanocytes and Melanoma Cells with Plant Extracts of theInvention.

Cell Culture:

In the report below, the starting material used

-   -   for L-18, is a powder (put in aqueous mother solution, then        adjusted to the indicated concentration),    -   for L-19, is a solution in a water/glycerol mixture at dry        extract 30%,    -   for L-20, is an aqueous solution at dry extract 5.6%,    -   for L-21, is an aqueous solution at dry extract 6%,    -   for L-22, is an aqueous solution at dry extract 15%,    -   for L-23, is a powder (put in aqueous mother solution, then        adjusted to the indicated, concentration),

Assessment of doses: Fully-matched epidermal melanocytes EM andepidermal keratinocytes (EK) were seeded into 6-well plates inserum-supplemented full MEM melanocyte and K-SFM keratinocyte medium for24 h. The cells were switched to serum free medium (so-called starved)supplemented with L-18 (0.001-0.005%), L-19 (0.5-1.0%), L-20 (0.1-0.5%),L-21 (10 μg/ml), L-22 (0.1-1.0%) and L-23 (0.01-0.05 μg/ml) for 12 and72 h. Controls included IBMX (100 μM) and niacinamide (10 μM).Cytotoxicity was assessed by cell death and cytopathologic change inmorphology.

Approximately 1×10⁴ EM and 2×10³ FM55 melanoma cells were seeded intoeach well of a Lab-Tek® 8-well chamber slide and allowed to attach for24 h. Cells were then washed 3-times with sterile PBS and supplementedwith 350 μl of either fresh serum-free RPMI medium (melanoma cells) orstarved serum-free and BPE-free melanocyte medium (EM) and incubated at37° C. and 5% CO2 for 24 h. Cells were then washed with sterile PBS3-times and incubated with IBMX 1×10⁴ M at 37° C. and 5% CO2 for 12, 24,and 72 h. Cells were then gently washed 3-times with sterile PBS andfixed in ice-cold methanol for 10 minutes at −20° C. Slides were storedat −20° C. until immunocytochemistry was performed.

Melanin Assay: 500 μg/ml of synthetic melanin (Sigma, UK) was preparedin 1 M sodium hydroxide (NaOH) (BOH Ltd, UK) and dissolved in asonicating water bath for 20 minutes. From this stock solution, variousmelanin standards were prepared in 1 M NaOH from 50 μg/ml to 1 μg/ml.The melanin standards were pipetted into a 96 well plate to produce acalibration curve for the assessment of melanin content in the testsamples. 400 μl of 1 M NaOH was added to each cell pellet and dissolvedon a heat block (100° C.) for 15 minutes. The pellets were vortexedvigorously and the solibulized pellet was pipetted into the same 96-wellplate. The optical densities of the sample were read at 495 nm on aDYNEX REVELATION 4.02 program. Melanin content of each test sample wasread from the calibration curve.

DOPA oxidase detection using non-denaturing SDS-PAGE for assessment oftyrosinase activity: Approximately 5×10⁵ of EMc, and FM55 melanoma cellswere seeded into three T75 flasks and were incubated at 37° C. and 5%CO2 overnight. The cells were prepared for SDS-PAGE and transblottedonto PVDF membranes. 70 μg of un-reduced protein extract without boilingwas pipetted into the appropriate wells of 8% SDS-PAGE gels. The PVDFmembrane containing the separated proteins was washed once in 1×PBS andthen incubated at RT in 5 mM L-DOPA in 0.1 M sodium phosphate buffer for3 hours with three changes of the L-DOPA. The L-DOPA reaction wasstopped by washing the membrane in distilled water and the membrane wasscanned.

Western blot analysis: EMc in a confluent T225 flask were trypsinisedand seeded into T25 flask with full medium and allowed to attachovernight. 24 h before treatment the medium was replaced with starvedmedium for 24 h. Cells were washed 3-times with sterile PBS, andincubated in starved medium alone, or L-18 (0.001%), L-19 (1%), L-20(0.5%), L-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml) for 12 and 72h. Controls included IBMX (100 μM) and niacinamide (10 μM).

40 μg of total protein from each cell extract was electrophoresed inreducing SDS-8%-PAGE and blotted on PVDF membranes (MilliporeCorporation, Bedford, Mass.). The membranes were blocked with 5% milkPBS/0.075% Tween 20 for 2 h at room temperature and were then probedwith primary antibodies for overnight at 4° C. The molecular weightladder (Magik Marker, Invitrogen) was incubated in 5% milk PBS/0.075%Tween 20 and the membrane strips were incubated with either 1 ml of 5%milk PBS/0.075% Tween 20 (negative control), or 1 ml of rabbitanti-Myo-X polyclonal antibody (1:200) in 5% milk PBS/0.075% Tween 20),1 ml of rabbit anti-fascin (1:500) polyclonal antibody in 5% milkPBS/0.075% Tween 20) and 1 ml of goat anti-actin (1:1000) polyclonalantibody in 5% milk PBS/0.075% Tween 20) on a rocking platform overnightat 4° C.

After extensive washes the blots were incubated with horseradishperoxidise conjugated secondary antibodies for 2 h. The molecular weightladder was then incubated with 1 ml of HRP-goat anti-human IgG (H+L)(Zymed, USA) (1:500) diluted in 5% milk PBS/0.075% Tween 20 and membranestrips were incubated in 1 ml of anti-rabbit IgG, horseradish peroxidaselinked whole antibody (HRP) secondary antibody (Amersham Biosciences,UK) (1:700 diluted in 5% milk PBS/0.075% Tween 20) on a rocking platformfor 2 hours at room temperature. The washing procedure was repeated andthe membrane strips were incubated in LumiGLO® Reagent and Peroxide(BioLab Ltd, UK) for 2 minutes at room temperature. The chemiluminescentsignal was detected by exposing the blot strips to Kodak XRA X-ray film(Kodak, UK) at various exposure times, followed by development indeveloping solution (Kodak, UK) until bands appeared, rinsed in tapwater, fixed in the fixer (Kodak, UK) until the film turned blue, thenrinsed with tap water and allowed to dry. The membrane was then labelledand scanned densitometric analysis a software (Image Master Total labversion 1.11).

Immunofluorescence staining: For EM:KC co-culture studies fully matchedmelanocytes (p4) and keratinocytes (p2) were seeded onto 8-well Lab-Tek®chamber slides at a cell density of 1×10⁴ cells/well and in a ratio of 1melanocyte to 10 keratinocytes. Co-cultures were maintained overnight(16 h) in a mixture of full K-SFM and MEM (co-culture medium) to allowcell attachment, followed by medium replenishment for a further 24 h.Cells were then fixed in ice-cold methanol for 10 min at −20° C., washedin PBS and then blocked with 10% donkey serum for the detection ofprotein expression in filopodia.

For double labelling experiments the first primary antibody, Myo-X(1:100) (Santa Cruz, Calif., USA) was applied overnight at 4° C.,followed by incubation with FITC-conjugated secondary antibody (1:100)for 1 h at room temperature. The second primary antibody, eithercytokeratin (1:100) (Abcam, Cambridge, UK) or β-Actin (1:100) (SantaCruz, USA) or Fascin (1:100) (Santa Cruz, USA) or NKi/beteb (1:30)(Monosan) was applied for 1 h at room temperature followed by aTRITC-conjugated secondary antibody (1:100) (Jackson ImmunoresearchLaboratories, Inc., West Grove, USA). DAPI (Vector Laboratories,Burlingame, Calif.) was used to stain nuclei. Images were captured witha cooled Hamamatsu digital camera using a 100× objective andpost-processed using Paint Shop Pro (Jasc Software Ver. 7. CA, USA).Negative controls included the omission of primary antibody andreplacement with non-immune serum from secondary antibody host andinclusion of secondary antibodies.

Quantitative analysis of melanosome transfer: Fully-matched epidermalmelanocytes (p4) and epidermal keratinocytes (p2) from 4 normalindividuals (i.e., F39; F67; F54; F52) were maintained in 8-wellLab-Tek® chamber slides at a total cell density of 2×10⁴ cells/well andin a ratio of 1 melanocyte to 10 normal keratinocytes. The co-culturewas maintained for further 24 h before washing 3-times with sterile PBSbefore incubation with starved medium alone, or L-18 (0.001%), L-19(1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml) for12 and 72 h. Controls included IBMX (100 μM) and niacinamide (10 μM).Cells were then processed for immunofluorescence staining with gp100 toassess melanosome transfer to keratinocytes.

Evaluation of melanosome transfer was performed by counting fluorescentgp100-positive spots within recipient keratinocytes in 5 randommicroscopic fields per well at 100× magnification (oil-immersion) in 3independent experiments. To avoid counting melanin granules that maystill be associated with melanocytes, we only counted gp100-positivespots within keratinocytes that were not in direct contact withmelanocytes.

Knockdown of Myo-X using siRNA: A synthetic siRNA targeting human Myo-X(5′-CAGCGGTATAAGAGAAATCAA-3′) (SEQ ID No 1) and a non-silencing control,consisting of siRNA that has no known homology with mammalian genes(5′-AATTCTCCGAACGTGTCACGT-3′) (SEQ ID No 2) was obtained from Qiagen,Valencia, Calif. A day before siRNA treatment, 5×10⁵ epidermalmelanocytes per well were plated onto six-well plates at 50-60%confluency and incubated at 37° C. for 12 h. Cells were then treated for12 h with a final concentration of 25 nM siRNA by using HiPerFectTransfection Reagent (Qiagen, Valencia, Calif.) following themanufacturer's instructions. Fluorescence microscopy was used to verifythat approximately 100% of the cells (using non-silencing controllabelled with alexa fluor 488 labels) had taken up the siRNA. At 12 h,the cells from each well were replated into 8-well Lab-Tek® chamberslides. For co-culture studies siRNA treated melanocytes were seededwith normal keratinocytes (passage 2) in 8-well Lab-Tek® chamber slidesat a cell density of 2×10⁴ cells/well and in a ratio of 1 siRNA treatedmelanocyte to 10 normal keratinocytes. At 24 h the co-culture wereprocessed for double Immuno fluorescence staining with gp100 andcytokeratin or Myo-X to study of the influence of gene knockdown onmelanosome transfer, and parallel samples were assayed byimmunofluorescence staining and western blot analysis to verify geneknockdown. For another experiment these co-culture were treated with orwithout L-18-23 for 24 h.

Results/Discussion

Dose Selection of Plant Extracts L-18-23:

Cells were incubated with the plant extracts and modulators ofpigmentation (IBMX and niacinamide) at a range of concentrations. Somedoses resulted in cell death or abnormal change in cell morphology(e.g., vacuolation). The inventor showed that optimal L-18-23 activedoses that were not associated with altered cell growth or morphology.These doses were therefore used for the remainder of the study.

Normal human epidermal keratinocyte culture (Female-67; p2) were treatedfor 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml),L-22 (1%) and LVMH-23 (0.05 μg/ml). Controls: Melanocytic modulators:IBMX (100 μM) and niacinamide (10 μM).

Normal human epidermal melanocyte culture (Female-67; p4) were treatedfor 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml),L-22 (1%) and L-23 (0.05 μg/ml). Controls: Melanocytic modulators: IBMX(100 μM) and niacinamide (10 μM).

Dose Selection for Experimental Design

Final dose selection for study on MC- Plant Extracts KC Co-culture L-180.001%  L-19 1.0% L-20 0.5% L-21   10 μg/ml L-22 1.0% L-23 0.05 μg/mlEffect of Plant Extracts L-18-23 on Melanogenesis:

Normal human epidermal melanocytes (F52; p5) and human melanoma (FM55)were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21(10 μg/ml), LVMH-22 (1%) and L-23 (0.05 μg/ml). In addition IBMX andNH4Cl (positive melanocytic modulators) and niacinamide (negativemodulator) were used as controls. Visible change was not particularlyevident in normal melanocyte due to their already high basal melaninlevels (FIG. 1). By contrast, visible change was evident in melanomacells basal melanin levels were low (FIG. 2). L-19, L-20, L-21 and L-23significantly reduced melanin content compared with basal levels in bothnormal melanocytes and FM55 melanoma cells. L-22 however was associatedwith an increase in melanin content compared with basal levels. L-18 didnot induce a significant change in these cells. Results are summarizedin Table 1 and in FIGS. 1 & 2.

FIG. 1: Effect of Plant Extracts L-18-23 Actives on Melanogenesis inNormal Pigmented Human Melanocytes

Normal human epidermal melanocytes (Female-52; p5) were treated for 72 hwith L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%)and L-23 (0.05 μg/ml). Controls: Melanocytic modulators: IBMX (100 μM),NH4Cl (100 μM), and niacinamide (10 μM).

(A) Melanin content was determined spectro-photometrically (475 nm)after sodium hydroxide solubilise

(B) Visible change was not particularly evident in these cells due totheir already high basal melanin levels. Results were expressed aschange in melanin content (pg/cell) compared to unstimulated controllevels. Means are ±SEM of 3 independent experiments with *P<0.05,**P<0.01, ***P<0.001. NS—Not SignificantFIG. 2: Effect of Plant Extracts L-18-23 on Melanogenesis inModerately-Pigmented FM55 Melanoma

FM55 cells were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20(0.5%), LVMH-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml). Controls:Melanocytic modulators: IBMX (100 μM), NH4Cl (100 μM), and niacinamide(10 μM).

(A) Melanin content was determined spectrophotometrically (475 nm) aftersodium hydroxide solubilisation. Cells with low basal melanin levelsshowed visible increases in melanogenesis after IBMX and NH4Clstimulation.

(B). Results were expressed as the change in melanin content (pg/cell)compared to unstimulated control levels. Means are ±SEM of 3 independentexperiments with *P<0.05, **P<0.01, ***P<0.001. NS—Not Significant.

TABLE 1 Changes in melanin levels after incubation with plant extractsL-18-23 Basal NC IBMX NH₄Cl L-18 L-19 L-20 L-21 L-22 L-23 F52 EM %change 0 −4.01 +74.57 +166.17 −4.57 −34.43 −15.66 −43.58 +31.60 −25.25(pg/cell) (35.63) (34.2) (62.2) (94.83) (34.04) (23.36) (30.62) (20.2)(46.89) (26.62) FM55 % change 0 −5.94 +250.8 +478.82 −3.22 −21.90 −20.36−58.65 +29.33 −33.20 (pg/cell) (9.92) (9.33) (34.8) (56.83) (9.62)(7.74) (7.92) (4.2) (12.82) (6.63)Effect of Plants Extracts L-18-23 on Tyrosinase Activity:

Normal human epidermal melanocytes (F52; p5) and human melanoma (FM55)were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21(10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml). In addition IBMX and NH4Cl(positive melanocytic modulators) and niacinamide (negative modulator)were used as controls. IBMX and NH4Cl were associated with a dramaticincrease in dopa oxidase activity of tyrosinase in both EM and FM55cells (FIGS. 3 & 4). Of the L-18-23, L-19, L-20, L-21 and L-23significantly reduced tyrosinase activity in both normal melanocytes andFM55 melanoma cells compared with basal levels (FIGS. 3 & 4). Bycontrast, L-22 however was associated with a significant increase intyrosinase activity compared with basal levels, while L-18 did notinduce any significant change. These results are summarized in Table 2.

FIG. 3: Effect of Plant Extracts L-18-23 on Tyrosinase Activity inPigmented Normal Human Melanocytes Normal human epidermal melanocytes(Female-52; p5) were treated for 72 h with L-18 (0.001%), L-19 (1%),L-20 (0.5%), L-21 (10 μg/ml), LVMH-22 (1%) and L-23 (0.05 μg/ml).Controls: Melanocytic modulators: IBMX (100 μM), NH4Cl (100 μM), andniacinamide (10 μM).(A) Protein extracts were electroblotted and membranes stained withL-DOPA for the estimation of tyrosinase activity.(B) Densitometric scanning of band intensities and values were expressedas a fold increase compared to unstimulated control levels Means are±SEM of 3 independent experiments with *P<0.05, **P<0.01, ***P<0.001.NS—Not Significant.FIG. 4: Effect of Plant Extracts L-18-23 on Tyrosinase Activity inModerately Pigmented FM55 Melanoma.

FM55 cells were treated for 72 h with L-18 (0.001%), L-19 (1%), L-20(0.5%), L-21 (10 μg/ml), L-22 (1%) and L-23 (0.05 μg/ml). Controls:Melanocytic modulators: IBMX (100 μM), NH4Cl (100 μM), and niacinamide(10 μM).

(A) Protein extracts were electroblotted and membranes stained withL-DOPA for the estimation of tyrosinase activity.

(B) Densitometric scanning of band intensities and values were expressedas the fold increase compared to unstimulated control levels. Means are±SEM of 3 independent experiments with *P<0.05, **P<0.01, ***P<0.001.NS—Not Significant.

TABLE 2 Changes in tyrosinase activity after incubation with plantsextracts L-18-23 Basal NC IBMX NH₄Cl L-18 L-19 L-20 L-21 L-22 L-23 F52EMc % change 0 −4.58 +72.47 +159.63 −6.17 −55.23 −13.76 −60.55 +16.50−39.44 FM55 % change 0 −3.31 +141.72 +408.27 −5.29 −50.37 −22.15 −42.54+43.54 −42.88

However, results from EM as monoculture may not fully reflect paracrineinfluences from keratinocyte and thus may not fully reveal L-18-23associated effects. Therefore, the above assessment for tyrosinaseactivity was repeated with two of the plant extracts; one which showedno change (L-18) and one with a significant decrease (L-23) infully-matched EM:KC co-cultures. It was interesting to note that underco-culture conditions, L-18 now showed a significant decrease in dopaoxidase activity of tyrosinase (from −3.46% when on EM cells grown aloneto −57.02% when co-cultured with matched KC). For L-23, a furtherdecrease in dopa oxidase activity of tyrosinase was seen when inco-culture (i.e., from −41.16% in monoculture to −76.17% in co-culture)(FIG. 5, and Table 3). A similar positive and negative trend was alsoseen with IBMX and niacinamide respectively.

FIG. 5: Effect of Plant Extracts L-18 and L-23, IBMX and Niacinamide onTyrosinase Activity in Normal Human Melanocyte and KeratinocyteCo-Culture (Note: Protein Loading was Normalized for Melanocyte ProteinOnly).

Normal human epidermal melanocyte culture (Female-52; p5) and matchedco-culture F52 MC-KC were treated for 72 h with L-18 (0.001%), L-23(0.05 μg/ml). Controls: Melanocytic modulators: IBMX (100 μM), andniacinamide (10 μM). Negative control lane: Keratinocytes only

(A) Protein extracts were electroblotted and membranes stained withL-DOPA for the estimation of tyrosinase activity.

(B) Densitometric scanning of band intensities and values were expressedas the fold increase compared to unstimulated control levels. Means are±SEM of 3 independent experiments with *P<0.05, **P<0.01, ***P<0.001.

TABLE 3 Changes in EM tyrosinase activity on EM:KC co-cultures afterincubation with plants extracts L-18 and L-23 Basal EM- NC L-18 L-23IBMX EM EK EM EM-EK EM EM-EK EM EM-EK EM EM-EK % Change in EM Tyrosinase0 +12.16 −2.12 −20.50 −3.46 −57.02 −41.16 −76.17 +56.51 +93.41 Activityvs Basal EM level (i.e. 0) % Change in EM Tyrosinase +12.16 −18.84−56.47 −57 +23.57 Activity of stimulated EM vs stimulated EM-EK) level(i.e. 12.16)Quantitative Analysis of gp100-Positive Melanosome Transfer Using PlantsExtracts L-18 and L-23:

Gp100 immunostaining provides a powerful tracking method for the globalassessment of melanin transfer to keratinocytes, and for the evaluationof melanocyte phenotype modulators (Singh et al., 2008). Doubleimmunolabelling (NKi/beteb and anti-cytoskeleton antibody) of the EM:KCco-cultures after treatment with all L-18-23, a phosphodiesteraseinhibitor IBMX (i.e., cAMP inducer) and niacinamide all revealed clearchanges in the number of fluorescent green gp100-positive melaningranules in keratinocytes (FIG. 6). In this assay melanosome transferfrom melanocytes to matched keratinocytes under basal (i.e.,unstimulated) conditions was determined at an average of 27.3gp100-positive spots per keratinocyte. However, this was increasedalmost 4-fold after 24 h stimulation of co-cultures with IBMX to 99.5gp100-positive spots per keratinocyte. Conversely, niacinamide reducedmelanosome transfer by 28% (19.6 gp100-positive spots per keratinocyte;p<0.01; a result that correlates with its clinical use as a lightener ofcutaneous pigmentation (Hakozaki et al, 2002; Greatens et al., 2005).

L-18, L-19, L-20, L-21 and L-23 all significantly reduced melanosometransfer as evidenced by a reduction of melanosome transfer compare tounstimulated control. By contrast, an increase in melanosome transfer tokeratinocytes was observed when co-cultures were stimulated with L-23),compared with unstimulated basal levels.FIG. 6: Quantitative Analysis of Melanosome Transfer after Incubationwith Plants Extracts L-18-23 (Table 4)

Melanocyte-keratinocyte matched co-cultures (Female-39) were treated for24 h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22(1%) and L-23 (0.05 μg/ml) along with parallel known melanocyticmodulators (IBMX (100 μM) and niacinamide (10 μM).Double-immunolabelling with anti-gp100 antibody (green) and anti-actinantibody (red) revealed clear changes in the number of fluorescent spotstransferred to keratinocytes. Quantification of transferred melanosomestaken from 5 randomly-selected microscopic fields (total 20 cells perfield) for each of the different treatment groups. Values were expressedas the percentage increase in the number of gp100-positive spots perkeratinocyte compared to unstimulated control levels. Means are ±SEM of4 independent experiments with **P<0.01, ***P<0.001.

TABLE 4 gp100⁺ Spots/KC 27.3 ± 0.9 99.5 ± 5.4 19.6 ± 0.7 17.8 ± 1.5 16.5± 1.9 21.7 ± 0.8 17.6 ± 0.9 36.0 ± 2.9 14.5 ± 2.2 % Change +264 .28 .34.39 .20 .35 +31 .47 of Melanin Transfer vs BasalEffect of Plant Extracts L-18-23 on Myo-X Expression in Normal HumanMelanocytes.

Double immunolabeling with antibodies against the Myo-X (Date not shown)and gp100 revealed prominent Myo-X expression in EM cell periphery anddendritic tips. Myo-X was also detected in EM filopodia. We have foundthat IBMX increased the expression of Myo-X and the overall number offilopodia (as suggested by total number of yellow spots beyond EMdendritic tips). IBMX also enhanced Myo-X localization in EM filopodiacompared with untreated cells in. By contrast, Myo-X expression wassignificantly down-regulated in EM by L-18, L-19, L-20, L-21 and L-23,while it was upregulated by L-22 alone. The localization of Myo-X withinEM filopodia was also significantly reduced by these plant extracts.

Western blot analysis of EM (F39) stimulated for 12 h by IBMX (100 μM)showed a 3-fold increase in Myo-10 expression as compared with theuntreated controls (FIG. 7A,B). By contrast, the melanosome transferinhibitor niacinamide (10 μM) reduced Myo-X expression by 60%. AllL-18-23 significantly down-regulated EM Myo-X expression (FIG. 7A,B),with the exception of L-22.

FIG. 7: Effect of Plant Extracts L-18-23 on Myo-X Protein Expression byWestern Blotting in Normal Human Melanocytes

Normal human epidermal melanocyte cultures (F39-EM) were treated for 12h with L-18 (0.001%), L-19 (1%), L-20 (0.5%), L-21 (10 μg/ml), L-22 (1%)and L-23 (0.05 μg/ml). Controls: IBMX (100 μM) and niacinamide (10 μM).

(A) Cell extracts were analyzed by Western blotting using anti-Myo-X andanti-β-actin as a loading control.

(B) Densitometric analysis of Myo-X and actin band intensities expressedas percentage change compared to unstimulated controls. Means are ±SEMof 3 independent experiments with **P<0.01, ***P<0.001.

The Knockdown of Myo-X Expression in Epidermal Melanocytes InhibitsMelanosome Transfer to Epidermal Keratinocytes

The role of Myo-X in melanosome transfer was investigated by usingsynthetic siRNA for 12 h against human Myo-X and non-silencing controlsiRNA. Myo-X silencing in melanocyte cells was assayed by western blotanalysis. This inhibition was also confirmed by immunofluorescence,which also showed that the expression of Myo-X was almost completelyinhibited compared to the non-silencing control siRNA.Double-immunolabelling of the Myo-X-silenced melanocytes with anti-gp100antibody revealed the absence of filopodia as evidenced by the lack ofgp100-positive melanosome around the melanocyte tips. Melanocyte treatedwith non-silencing control siRNA showed prominent Myo-X and gp100 in thefilopodial region.

We were keen to determine, whether endogenous Myo-X expression is arequirement for melanosome transfer via filopodia in melanocyte. Inorder to test the involvement of Myo-X in melanin transfer,Myo-X-silenced melanocytes and non-silenced melanocytes were used toestablish the MC-KC co-culture for 24 h. Double Immunolabelling of thisco-culture with anti-gp100 antibody and anti-Myo-X clearly showed theinhibition in the number of fluorescent spots transferred tokeratinocytes cultured with Myo-X-silenced melanocytes.Double-immunolabelling with anti-gp100 antibody and anti-cyokeratinrevealed clear changes in the number of fluorescent spots (representingmelanin granule) transferred to keratinocytes.

Using this assay the rate of melanosome transfer from melanocytes tokeratinocytes with non-silencing control siRNA conditions was determined(FIG. 8). This level of melanosome transfer was reduced by 80% inMyo-X-silenced melanocytes cultured with normal keratinocyte cells for24 h (FIG. 8). This is the first demonstration of a physiological roleof Myo-X in the successful transfer of melanosomes from melanocytes tokeratinocytes.

In the following examples, the percentages are given by weight from thetotal weight of the composition. The amount of plant extracts isexpressed as dry weight.

EXAMPLE 1 Cosmetic Powder for Lightening the Facial Complexion

Cyathea cumingii leaf extract  0.5% Microcellulose 20.0% Sodium laurylsulphoacetate 15.0% Fragrance, dyes, preserving agents qs Talc qs 100%

This powder allows cleansing of the skin, and also makes it possible,through regular use for a few days, to lighten the complexion. It can beapplied to the facial skin once or twice a day.

EXAMPLE 2 Depigmenting Cosmetic Day Cream in Emulsion-Gel Form

Secale cereale seed extract 0.1% Glycerol 5.0% caprylic/capric/succinictriglycerides 5.0% Octyl methoxycinnamate 1.0% Copolyol dimethicone 0.5%Acrylates/C10-30 alkyl acrylate crosspolymer 0.5% Neutralising agent asneeded. Preservation agents, odorant, colouring agents as needed. Waterqs 100%

Some individuals subjected to more or less intense irradiation due todaylight, or even direct sunlight, would like to keep a light skin andavoid the appearance of pigmenting spots.

The use of the emulsion-gel defined above provides the means ofachieving this purpose. This composition is usually applied on the facein the morning. It is equally effective for preventive and remedialaction on regular or irregular pigmentation of the face.

EXAMPLE 3 SPF 30 Protective Fluid Preventing Pigmentation Spots

Thassiora pseudonana extract 0.01%  Volatile pentacyclomethicone 49.0% Titanium dioxide 15.0%  Octyl methoxycinnamate 7.5% Glycerine 5.0%Phenyltrimethicone 5.0% Copolyol dimethicone 3.0% Polymethylmethacrylate2.5% Butyl methoxydibenzoyle methane 1.0% Neutralising agent, odorant,preservation agents, as needed. antioxydisers Water qs 100%

The protective fluid is used to prevent the appearance of pigmentationspots in persons subject to this phenomenon, before exposure to intensesolar radiation. It should be noted that the presence of a highconcentration in the solar filter compensates for the reduction innatural protection due to the drop in the melanin content.

EXAMPLE 4 Dermatological Cream for the Treatment of SkinHyperpigmentations of Pathological or Trauma-Based Origin

Cyathea cuminghii leaf extract 0.3% Glyceryl stearate + PEG-100 stearate5.0% Hydrogenated polyisobutene 4.0% Magnesium ascorbyl phosphate 3.0%Glyceryl tricaprylate/caprate 3.0% Squalane 3.0% Glycerol 2.0% Beeswax1.5% Cetearyl octanoate 1.5% Cetyl alcohol 1.0% Stearyl alcohol 1.0%Dimethicone 1.0% Xanthan gum 0.3% Ethylenediaminetetraacetic acid 0.2%Citric acid 0.1% Sodium citrate 0.1% Neutralizing agent, fragrance,preserving agents qs Water qs 100%

The use of this cream makes it possible to reduce skinhyperpigmentations of pathological or trauma-based origin. This creamalso makes it possible to reduce the colour contrast at the periphery ofdepigmented areas in the case of vitiligo.

EXAMPLE 5 Cosmetic Face Lotion for Lightening the Complexion

Thalassiosira pseudonana 0.01%  Ethyl alcohol 30.0%  PPG-3 myristylether 5.0% Glycerol 2.0% Carbomer 0.2% Polysorbate 20 0.2% Neutralizingagent, fragrance, preserving agents qs Water qs 100%

This lotion for lightening the complexion is used after removing make-upfrom the skin and cleansing the latter.

EXAMPLE 6 Cosmetic Facial Lightening Serum

Secale cereale (Rye) seed extract 0.2% Glycerol   2% Tetrasodium EDTACitric acid qs desired pH Trisodium citrate Xanthan gum 0.3%Polyacrylamide, C13.14 isoparaffin, laureth-7 0.5% Dimethicone copolyol0.3% Fragrance, dye, preserving agent qs Water qs 100%

A drop of this very concentrated serum composition is applied to theface, generally before the application of a face cream. This serum isnormally used as treatments of one to two weeks so as to obtain ormaintain a lightening of the complexion.

EXAMPLE 7 Cosmetic Lotion for Lightening Body Hair

Thalassiosira pseudonana extract 0.01%  Panthenyl ethyl ether 0.5%DL-α-tocopheryl acetate 0.2% Polysorbate 60   1% Fragrance 0.2% Glycerol0.5% Dye qs Water qs 100% Alcohol 50%

This lotion is applied to the areas with hair that are to be lightened,in particular the arms, for the amount of time sufficient to obtaingradual lightening of the hairs.

EXAMPLE 8 Cosmetic Anti-Spot Gel-Cream for the Hands

Caprylic/capric diglyceryl succinate   6% Octyl octanoate 2.5% Octylmethoxycinnamate   6% Cyathea cumingii leaf extract 0.3%Phenyltrimethicone 2.5% Benzophenone-3 0.5% Sodium hyaluronate 0.1%Xanthan gum 0.2% Acrylates/C10.30 alkyl acrylate copolymer 0.5% Glycerol  2% PEG 150   3% Neutralizing agents, dyes, fragrance, preserving qsagents Purified water qs 100%

This cream must be applied directly to the spots (solar and/or senilelentigo) on the hands, so as to reduce the colouration of said spots.

EXAMPLE 9 Dermatological Solution for Treating Pathological HyperPigmentation

Secale cereale seed extract 0.2% Volatile pentacyclomethicone 49.0% Titanium dioxide 15.0%  Octyl methoxycinnamate 7.5% Glycerine 5.0%Phenyltrimethicone 5.0% Copolyol dimethicone 3.0% Polymethylmethacrylate2.5% Butyl methoxydibenzoylmethane 1.0% Neutralising agent, Odorant,Preservation agents, as needed antioxidants Water Qs 100%

This serum is applied to the skin daily for the treatment of personssuffering from regional hyper pigmentation.

EXAMPLE 10 Hair Tonic Lotion for Depigmenting Hair

Secale cereale seed extract  0.2% 3-Methylxantine 0.03% Alcohol 30.0%Perfumed aqueous excipients with perfume solubilizer Qs 100.0%

EXAMPLE 11 Tanning Sun Cream

Hydrolysed soy flour (soy seed pericarp extract) 0.3% Isocetyl stearate8.0% Hydrogenated groundnut oil 10.0%  Lanolin oil 3.5% Cetyl alcohol5.0% Stearyl alcohol 2.5% Light liquid paraffin 10.0%  Neutralizedphosphoric acid monoester of 3.0% oxyethylenated cetyl alcoholOctylmethoxy cinnamate 5.0%

This phase is emulsified with an aqueous phase qs 100% containing:

Pantothenol 0.1% Preservatives 0.2%

EXAMPLE 12 Lotion for Strengthening Natural Sun Protection

Alcohol 42.50%  Propylene glycol 3.00% Menthol 0.05% Hydroxypropylmethyl cellulose  1.5% Hydrolysed soy flour  0.2% Perfumed aqueousexcipients Qs 100.0%

This lotion is applied locally, preferably twice a day, every day for 3to 8 days preceding prolonged exposure to the sun.

The daily applications can be continued during the period of exposure.

EXAMPLE 13 Hair Tonic Lotion for Pigmenting Gray Hair

Hydrolysed soy flour (soy seed pericarp extract) 0.10% 3-Methylxanthine0.03% Alcohol 30.0% Perfumed aqueous excipients with perfume Qs 100.0%solubilizer

This lotion can be applied to the hair and scalp twice a day for avigorous treatment intended for rapidly reducing the appearance of whitehair.

EXAMPLE 14 Dermatological Gel Intended for Promoting the Pigmentation ofthe Skin

Hydrolysed soy flour 0.10% Ethanol 0.03% Distilled water 30.0% Gellingexcipient, with 1.25% Carbopol 940 ® Qs 100.0% gel

REFERENCES

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The invention claimed is:
 1. A method of assessing the ability of asubstance to modulate the transfer of melanin for a melanocyte to akeratinocyte, the method comprising the steps of: a)—providing a testsubstance; b)—providing a cell expressing Myo-X; and c)—determining theability of the test substance to modulate the expression or theactivation of Myosin-X (Myo-X) in the cell.
 2. The method of claim 1,wherein the cell expressing Myo-X is a melanocyte or a keratinocyte. 3.The method of claim 1, comprising comparing the expression or activationof Myo-X in a cell treated with a test substance with the expression orthe activation of Myo-X in a control cell.
 4. The method according toclaim 1, wherein determining the ability of the test substance tomodulate the expression or the activity of Myo-X includes: measuring theamount of Myo-X protein in or on the cell; determining the amount of aprecursor in the synthesis of Myo-X protein; or measuring the melanosometransfer level in the cell.
 5. A method of selecting an active substancecapable to enhance skin or hair pigmentation comprising: assessing theability of said substance to modulate the transfer of melanin accordingto steps a) to c) of the method of claim 1, selecting the substancecapable to increase the expression or the activation of MyoX.
 6. Amethod of selecting a substance capable to reduce skin or hairpigmentation comprising: assessing the ability of said substance tomodulate the transfer of melanin according to steps a) to c) of themethod of claim 1, selecting the substance capable to decrease theexpression or the activation of MyoX.
 7. A method according to claim 1,wherein the assessed substance is a plant extract.